Back with another episode of The Biocast. This episode looks at the evidence for endotoxin in causing obesity and type II diabetes in humans.
The topic is also closely tied into liver conditions like NAFLD and NASH and potentially any other chronic inflammatory condition.
The pre-release for Patreon supporters has the references. USD 5 per month pledge gets access to that and most of the other patreon content, including the series on Light Therapeutics and the current series on Endotoxin Antagonists, things that lower plasma endotoxin and/or the inflammatory response. I’ll be looking at diet, supplements, lifestyle and pharmaceutical agents.
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Music by BoxCatGames
Energy intake is associated with endotoxemia in apparently healthy men.
Human review, plasma LPS correlation to diet. Caloric intake and fat associated
Endotoxemia Is Associated With an Increased Risk of Incident Diabetes
Human association diabetes LPS
Natural Antibiotics and Insulin Sensitivity
Plasma Lipopolysaccharide Is Closely Associated With Glycemic Control and Abdominal Obesity
Prospective observational human study of bariatric surgery and LPS
Reduction in endotoxemia, oxidative and inflammatory stress, and insulin resistance after Roux-en-Y gastric bypass surgery in patients with morbid obesity and type 2 diabetes mellitus.
Human study, bariatric surgery
A metagenome-wide association study of gut microbiota in type 2 diabetes
Human Study, microbiome analysis, bacteria
Gut microbiota: a potential new territory for drug targeting
UC , Crohns , prebiotic , probiotic , type 1 diabetes ,
Human review article
Gut microbiome and metabolic diseases
Review, human, obesity, liver
A core gut microbiome in obese and lean twins
Human twin observation bacteria
Comparative meta-analysis of the effect of Lactobacillus species on weight gain in humans and animals.
Review meta analysis probiotic effects on obesity humans and animals
Lipopolysaccharide activates an innate immune system response in human adipose tissue in obesity and type 2 diabetes
Human Tissue Study, association of LPS and T2D Diabetes
A Marker of Endotoxemia Is Associated With Obesity and Related Metabolic Disorders in Apparently Healthy Chinese
Human Study, LBP as a marker for obesity
The Association between Visceral Fat and Endotoxin.
The endocannabinoid system links gut microbiota to adipogenesis.
Review, Overview LPS – EC – Inflammation fat storage
Gut Microbiota and Nonalcoholic Fatty Liver Disease: Insights on Mechanism and Application of Metabolomics
Review, humans , liver, mechanisms
Role of Bile Acid Sequestrants in the Treatment of Type 2 Diabetes
Stress Induces Endotoxemia and Low-Grade Inflammation by Increasing Barrier Permeability
Review mechanisms human article
High Fat Intake Leads to Acute Postprandial Exposure to Circulating Endotoxin in Type 2 Diabetic Subjects
Postprandial Effect of a High-Fat Meal on Endotoxemia in Arab Women with and without Insulin-Resistance-Related Diseases
Increase in Plasma Endotoxin Concentrations and the Expression of Toll-Like Receptors and Suppressor of Cytokine Signaling-3 in Mononuclear Cells After a High-Fat, High-Carbohydrate Meal
The Role of the Gut Microbiota in Energy Metabolism and Metabolic Disease
Review, Overview, Human, Mechanisms
Orange juice neutralizes the proinflammatory effect of a high-fat, high-carbohydrate meal and prevents endotoxin increase and Toll-like receptor expression
Impact of gut microbiota on diabetes mellitus
Okay so let’s take a look at this first paper (18). This was a dietary survey of over 1000 random subjects in France. These people kept a three day food record and then a smaller sample of about 200 of them had their plasma LPS measured. They were looking to find out the relationship between plasma LPS and food intake. For some reason the 200 people selected to have their plasma endotoxin tested were all men. The reason for this isn’t made clear in the paper.
The study claims to have found no significant link between cardiovascular disease risk factors, carbohydrate and protein intakes with plasma LPS. However positive correlations were found with dietary fat intake. They performed another analysis and found that endotoxaemia was associated with total energy intake.
This paper also describes experiments in mice that were run looking at variations of high calorie diets and their effects on plasma endotoxin. They found that high calorie diet increased plasma endotoxin but that a high calorie high fat diet increase endotoxin more than a comparable high calorie high carbohydrate diet. The paper mentioned recent observation that young adult mice have 40% more total body fat than germfree mice fed the same diet.
The experimental rodent diet in this experiment were similar to some of the diet I spoke about in the previous podcast, that is, the high-fat diet was 72% Fat from corn oil and lard and 28% energy from protein. The high-fat high-carbohydrate diet was 35% fat from corn oil and lard, 37% carbohydrate, and 28% protein.I read previous studies where fat levels around that number is 37% were enough to significantly increase endotoxin, so perhaps labelling that a high calorie high-carbohydrate diet is a bit misleading at least to my mind where I would have assumed a much lower percentage of fat maybe around 10%.
This paper does show similar increases in endotoxin from similar increases in dietary fat, the high-fat diet will increase plasma endotoxin 2 to 3 times above controls. While this study shows a correlation with energy intake and plasma endotoxin in humans it really doesn’t have the ability to limit the variables to say that it is simply higher caloric intake increases endotoxin, so it could be and it probably is that higher caloric intake is associated with higher fat intake that is the cause of increased plasma endotoxin.
The paper references rodent studies mentioned in the previous podcast showing that plasma endotoxin causes insulin resistance, obesity and information through mediators like TNF alpha and interleukin-6.
The paper discusses how there is no consensus on how to measure plasma endotoxin concentrations, that the use different methods in the human’s and in the rodents.
So this next paper is looking at similar measures (20).
This paper started telling us about the associations with endotoxin, obesity and diabetes, it also notes that endotoxin is cleared from circulation mainly by high-density lipoproteins or HDL. First they carried a survey of over 7000 people from ages 25 to 74 in different areas of Finland. This was a questionnaire along with clinical examination including weight, height, blood pressure and blood tests. Prior to blood test there was a four hour fast and participants were asked not to eat heavy meals on that day.
They tested baseline factors including cholesterol measurements,HDL and LDL and blood glucose along with CRP. The participants were then separated into two groups for analysis one being a group with no diabetes and the second being the group with incident diabetes. Most of the metrics would be as you might expect relevant to the two groups the plasma endotoxin in the non-diabetic group was 61.06 compared to 77.03 in the diabetic group, an increase in over 25%.
To the paper noted in the conclusions that not only was endotoxaemia associated will increased risk of clinically incident diabetes but that that risk was independent of other known risk factors for diabetes, namely, glucose, lipid, and CRP levels along with body mass index, and also that it was independent of factors known to be associated with serum endotoxin like cholesterol, triglycerides and particularly HDL.
The next will look at is titled natural antibiotics and insulin sensitivity (21).
This study introduces a couple of things that I don’t think we’ve spoken about in either of the podcasts the first is bactericidal permeability increasing protein or, BPI. This is thought to be involved in the response to bacteria and its properties are thought to be anti-inflammatory. The study wanted to find it if bpi was linked to insulin sensitivity and glucose tolerance. They also tested LBP, which is lipopolysaccharide binding protein. LBP binds to endotoxin and is important in the immune response, is produced in the liver and serves to amplify the response to endotoxin by delivering it to the endotoxin receptors. So bpi and LBP compete for binding endotoxin but in terms of the response to endotoxin the inflammatory response they are antagonistic.
BPI is seen as anti-inflammatory whereas LBP was associated with inflammation.
So this study looked at circulating bpi in healthy people in people with glucose intolerance and in people with type II diabetes, to look at the relationship with circulating endotoxin and they looked at the effect of Metformin on circulating bpi in people with glucose intolerance. there were a list of factors that would lead to exclusion from the study and many of them are correlated with plasma endotoxin including liver and thyroid problems.
So the study found that bpi was lower among patients with type II diabetes, they found that circulating bpi was negatively correlated with LBP in control and type II diabetes subjects. Circulating bpi was associated with age in non-diabetic subjects. Bpi is negatively correlated with fasting and post-load glucose insulin and A1c. Bpi was positively associated with HDL, white blood cell and neutrophil counts.
Bioactive LPS was negatively correlated with bpi and positively correlated with LBP. They then tested the effect of Metformin on two small groups, eight on Metformin and 12 on placebo. They found that insulin sensitivity and plasma bpi increased only in the Metformin group.
With regard to the mention of natural antibiotics title the authors were referring to LBP and bpi, bpi being the more potent antibacterial agent.
Okay so the next study on the list is on the association with endotoxin and glycaemic control with abdominal obesity (22).
This was a prospective observational study looking at obese patients undergoing bariatric surgery compared to controls. Plasma endotoxin, HbA1c, adipose tissue and bacterial DNA in adipose tissue were tested, Along with CD14 and HDL. It was a relatively small study with 24 and 17 people in the groups. The surgery was a laparoscopic Roux-en-Y gastric bypass used for patients with a BMI above 50 kg/m².
In agreement with previous data baseline levels of endotoxin and CD14 were significantly elevated in obese subjects. CD 14 is involved in the detection and inflammatory response to endotoxin. One year after surgery plasma endotoxin was reduced but CD14 was not. Concurrent evaluations showed no reduction in endotoxin after a period of lifestyle intervention. Baseline levels of endotoxin were correlated with HbA1c and intra abdominal fat. Endotoxin was also positively correlated with fasting triglycerides, BP, and body mass index while it was negatively correlated with HDL.
One year after surgery there was a direct correlation with plasma endotoxin and glycaemic control measured by HbA1c, triglycerides also tracked with those two metrics however, BMI, HDL, cholesterol and blood pressure did not.Another metric to quantify was the bacterial DNA in the fat tissue close to the gut, the findings here were similar to previous findings in mice of increased bacterial DNA in the samples, in the rodent models this is seen to precede insulin resistance and type II diabetes.
In the conclusion the do state that the mechanism implicated in endotoxin reduction by bariatric surgery is unknown. They present two hypotheses, one that endotoxin increases in circulation because the gut is more permeable in obesity and to that endotoxin will be transported across the gut barrier with increased dietary fat.
The next paper looks at gastric bypass surgery again (23).
This looked at 15 adults with morbid obesity and type II diabetes undergoing bariatric surgery and measured endotoxin along with endotoxin receptors and inflammatory markers. The two sets of data were compiled after an overnight fast prior to the surgery on the day of the operation and again after an overnight fast 180 days post surgery.
They found that endotoxin was significantly decreased at 180 days, nuclear factor kappa B binding was significantly decreased, NFKB is a pro-inflammatory transcription factor. CD 14 was decreased also along with toll -like receptor four and the like receptor two, both involved in sensing endotoxin and starting the inflammatory response. C-reactive protein was significantly decreased 180 days after the surgery. So this study seems to indicate a decreasing endotoxin following the surgery along with a decrease in markers of endotoxin detection and a reduction in inflammation as measured by C-reactive protein. Other pathological markers that are known to be associated with obesity, and type II diabetes were improved by the surgery, that is lowered free fatty acids and markers of non-alcoholic liver disease, steatosis, inflammation, and fibrosis.
The next paper and going to look at is and analysis of gut microbiota sampling from over 300 people looking at its relationship to type II diabetes. This study was performed with a Chinese population separated into diabetic and normal controls. So the first thing that they found was that type II diabetes was strongly associated with gut dysbiosis. They Noted a reduced population of butyrate producing bacteria, butyrate is a short chain fatty acid produced by bacterial fermentation that has many differing effects on the intestinal system most often cited as providing fuel for some of the cells in the GI system. The paper also reports that the analysis of bacterial gene functions indicates an increase in gut oxidative stress which itself is another indicator of or correlation with type II diabetes. the last thing on from this study is that they claim the analysis of gut metagenomic markers are more accurate at identifying type II diabetes than analysis of human genome.
The paper that I’m going to look at next discusses targeting the gut microbiota with drugs. It talks about how the gut flora play a role in drug metabolism and gives an example of radix ginseng which is metabolised by bacteria into a compound that exhibits significant anti-tumour and anti-allergic properties. The paper mentioned how a high-fat diet will alter microbial metabolism and affect the conversion of choline, potentially creating choline deficiency and causing non-alcoholic fatty liver disease. There is a table in the paper of a number of diseases and associations with imbalanced microbiota along with evidence of improvement from targeted drug therapy. For example ulcerative colitis is associated with an abnormal immune response to bacteria increased microorganisms in general but reduced populations of bifido and lactobacilli, in this case short-term benefits were observed with antibiotic or probiotic intervention.
The next paper that will look at is called a core gut microbiota in obese and lean twins (27). These sets of twins had one twin with a BMI above 30 and one with a BMI between 18.5 and 24.9. This paper analyses faecal microbial communities of adult female twins both monozygotic and dizygotic and their mothers, a total of 154 individuals analysed. The results show families share similar microbiology but there are individual differences, obesity was found to be associated with differences in bacteria and a reduction in bacterial diversity.
The next paper is an analysis of previous studies on the effect of lactobacillus on weight gain in humans and animals (28).
So this was specifically on lactobacillus containing probiotics in clinical studies in experimental models. 17 random controlled trials in humans were used, 51 studies on farm animals and 14 experimental models. They found that different Lactobacillus species were associated with different effects on weight regulation. Lactobacillus acidophilus was associated with significant weight gain in humans and animals whereas lactobacillus fermenting and lactobacillus ingluviei were associated with weight gain in animals. Lactobacillus plantarum showed weight loss in animals, while Lactobacillus gasseri was associated with weight loss in obese humans and in animals. The study also mentions the association with bifido bacterium and leanness.
So the next he looks at endotoxin and the innate immune system response in adipose tissue in obesity and type II diabetes (29).
To this paper looks at inflammatory markers and endotoxin markers in abdominal adipose tissue from lean non-diabetic subjects and obese non-diabetic subjects as well as type II diabetic subjects. They took samples of tissue and then treated the samples with refined endotoxin. The results showed that the adipose tissue is an important site of innate immune activation and that endotoxin can trigger diabetogenic pro-inflammatory cytokines. It also showed increased expression of the innate immune system from samples of subjects with obesity and type II diabetes. Notably MYD 88 and NFkB.
The paper also discusses the use of TZD drugs, knowing that they have no known interaction with gut absorption or kuppfer cell function and inferred that insulin itself might be a factor in endotoxin absorption into the bloodstream and that TZD may have anti-inflammatory role. Further TZD is known to increase the bioavailability of HDL which is involved in endotoxin clearance and associated with lower cytokine production in the presence of endotoxin.
The paper also discusses potentially decreased transit time in type II diabetes leading to increased transport of endotoxin and correlation with bacterial overgrowth in the context.
Next up is a paper looking at the association between plasma lipopolysaccharide binding protein and metabolic disorders. (30)
This study took a look at 1000 Chinese people, with approximately half normal weight and half obese or overweight groups. It looked at measures including fasting glucose, lipid profile, LBP, CRP and interleukin-6 interleukin-6. This study found higher LBP levels in overweight or obese individuals. LBP was positively correlated with body mass index, waist circumference, blood pressure, total cholesterol, LDL, cholesterol, triglycerides, glucose, insulin, HOMA-IR, pancreatic enzymes and leptin. LBP was negatively correlated with HDL and adiponectin. plasma LBP was highly correlated with the inflammatory markers interleukin-6 and C-reactive protein.
The next paper is on the association between visceral fat and endotoxin (32).
The paper tells us that visceral fat accumulation is a risk factor for cardiovascular disease and diabetes, it is associated with inflammation and also that endotoxin is related to inflammation and atherosclerosis. The study took 40 subjects calculated abdominal fat and tested serum endotoxin. The results showed that serum endotoxin was positively correlated with visceral fat and recommended that further investigation should be implemented.
To the next paper looks at the effect of stress on intestinal permeability (36).
Goes through the basics of endotoxin in the plasma the activation of inflammatory mediators and pathogen detectors like toll -like receptors. We are told that increased plasma endotoxin is associated with depression, chronic fatigue syndrome, heart failure, type II diabetes, autism, non-alcoholic fatty liver disease and inflammatory bowel disease.
So the hypothesis put forward is that stress, being an increased metabolic demand leads to the organism responding by making the intestinal wall more permeable in order to increase the availability of water and energy.
LBP is involved in liver clearance of endotoxin. LBP can also deliver endotoxin to CD14 which leads to TLR4 activation and from their NFKB which stimulates inflammatory mediators like interleukin-6 TNF alpha and prostaglandin E2, a metabolite of polyunsaturated fatty acid arachidonic acid which increases inflammation and is strongly correlated with inflammatory diseases like rheumatoid and osteoarthritis. the paper also details that there are factors involved in restraining this inflammatory response, signalling inhibitors of TLR4, NFKB etc.
Papers on to detail a number of ways in which food and water can be passed through the gut barrier and into the rest of the body. Increased permeability is linked with rheumatoid arthritis, multiple sclerosis, irritable bowel disease, chronic fatigue syndrome, asthma, depression, and type I diabetes. I think we’ve seen a number of those already linked to plasma endotoxin.
The article tells us that stress activates the sympathetic nervous system and hypothalamic pituitary adrenal axis or HPA axis. This stress response increases the availability of energy minerals and water to try and meet the metabolic needs and adapt to the stress. Blood flow, stress hormones and renin angiotensin aldosterone are all mediated in the stress response.
In rodent models of restricted stress or swimming stress the intestinal permeability was increased throughout the whole gastrointestinal system. Maternal deprivation and thermal injury derived stress created ulcers, altered gastrointestinal motility and increased permeability.
They also mentioned that exogenous corticotropin releasing hormone administration increased permeability and that that could be limited by a mast cell stabiliser. corticotropin releasing hormone is a peptide and neurotransmitter involved in the stress response, secreted by the hypothalamus and synthesised in peripheral tissues that has associations with Alzheimer’s and depression. CRH then stimulates the other stress-related factors like ACTH, beta endorphin, cortisol and glucocorticoids. CRH seems in some way to be involved in stress induced permeability of the gastrointestinal tract probably involving TNF alpha and interferon-gamma another cytokine involved in innate and adaptive immunity against bacterial infections associated with other inflammatory and autoimmune diseases.
The paper points out one of the possible adaptive purposes of these inflammatory mediators, that being redirecting energy rich nutrients towards immunity. Prostaglandins like PGE2 are involved in the initial inflammatory response to endotoxin, this prostaglandin was shown to directly stimulate the release of glucocorticoids from the adrenal gland in animals. One of the basic functions that glucocorticoids are involved with is in modulating glucose metabolism breakdown of muscle for sugar and breakdown of fat for fuel.Endotoxin stimulation of toll -like receptors in human adrenal cells result in the release of cortisol by way of PGE2.
The paper argues that in case of acute stress the responses is in order to increase the immune response to fight potential infection or prepare for a necessary fight or flight response. Referenced are rodent studies showing protection of the brain from information induced by acute stress by antibiotics or an inhibitor of MLCK. And both of those methods mitigated permeability and endotoxaemia the paper argues that this shows that it’s not the stress response but the immune response as a result of endotoxin increases due to permeability. Similar effects were seen with a bacterial probiotic, namely lactobacillus farciminis, which enhances the intestinal barrier function.
The authors make a point that they believe acute stress will not produce low-grade inflammation, arguing that new environmental multifactorial strategies such as the Western diet and psychosocial stress alter the gut microbiota and compromised barrier function in a previous study using a Western diet described as a high-fat diet and there are a number of studies showing that this does this regulate microbiota, increased permeability and low-grade endotoxaemia.
Another section in the article talks about the effect of prenatal stress on the microbiota of mice and monkeys lowering the effect of bifido and lactobacilli and how animals raised in germfree environments had exaggerated HPA axis response which could be normalised by the administration of bifidobacterium infantis.
The paper notes a lot of the data on high fat diet inducing endotoxaemia in humans and animals that we’ve already looked at. Wheat and other cereal grains are implicated in increasing permeability along with alcohol, and exercised induced heat stress. That is to say exercise increases body temperature and that reduces intestinal blood flow leading to an increase in permeability. This is mediated by activating the sympathetic nervous system and HPA axis. There are some studies that indicate certain types of lower-level activity may be beneficial in the context of lowering endotoxaemia which I might get to at a later point. The paper then wraps up by showing the associations with endotoxaemia and metabolic syndrome, inflammatory bowel disease, and psychiatric diseases or dementias like Parkinson’s or Alzheimer’s. That’s something I have pencilled in for a future podcast.
Ok, so the next a look at investigates high fat intake and its effect on circulating endotoxin in type II diabetics. (40)
This is a paper from the American diabetes Association published in 2012. The aim of this study was to determine circulating endotoxin after a high saturated fat meal. So they took 54 humans, and after an overnight fast gave them what they called a high-fat meal and tested some blood markers before and after.
The meal was 75 g of fat, 5 g of carbohydrate, and 6 g of protein.The high-fat meal was comprised of whipping cream. If we look at the nutritional statistics of whipping cream to get 75 g of fat, 46 of those grams are from saturated fat 21 arc from monounsaturated and 2.7 are from polyunsaturated.
So the study found that baseline endotoxin was higher in people with type II diabetes or impaired glucose tolerance versus controls. The levels were over 60% higher in Type II diabetics versus controls. The ingestion of high-fat meal led to significant increase in endotoxin, and four hours after the meal the type II diabetes group had higher circulating endotoxin than normal controls.
Next is another study on the effects of high fat meals on endotoxemia and metabolic markers (37).
So this study was looking at the effect of a high-fat meal on endotoxaemia in Arab women with and without insulin resistance related diabetes. They were split into three groups labelled non-diabetic, overweight, and type II diabetes. After an overnight fast of more than 12 hours they were giving the high-fat meal with a similar composition to the previous high-fat meal used in human studies are 75 g of fat 5 g carbohydrate 6 g of protein.
Measurements and fasting blood glucose lipids and endotoxin were measured every hour after the meal for four hours. Prior to the meal plasma endotoxin was significantly higher in overweight plus and type II diabetes groups.
In response to the high-fat meal the type II diabetes group had higher blood glucose immediately after meal which became lowest after four hours whereas the control and overweight plus groups had no significant changes.
The type II diabetes group had significantly higher triglyceride levels compared to the other two groups in this peaked in the 3 to 4 hour range.
In terms of the endotoxin levels they peaked at our four in all groups. Both overweight and type II diabetes groups had significantly higher levels than the controls. In all groups insulin peaked during hours one and two dropped in hours three and four. the type II diabetes group had significantly higher insulin than the other two groups.
Endotoxin was associated with LDL in all groups three hours after the meal. Endotoxin was associated with triglycerides 3 to 4 hours after the meal in people with type II diabetes. Endotoxins associated with triglycerides in the overweight plus group at baseline. In the control group associations were observed at all.
So overweight and type II diabetes groups started off with about twice as much circulating endotoxin as the control, the actual increase in endotoxin across all the groups from our to hour four was largest in the control group. The control group went from 1.5 to 2.1, the overweight group went from 3.0 to 3.5, the type II diabetes group went from 3.4 to 3.6 on average though the variability was also much higher in the overweight and type II diabetes groups.
The next paper looks at various meals and the response to plasma endotoxin and inflammatory markers along with reactive oxygen species (38).
Okay so this study took two groups with 10 people in each group all with healthy normal body mass index of less than 25. The researchers had them fast overnight and then took a baseline blood sample. Then one group was given a high-fat high carb meal of egg muffin sausage muffin sandwiches and two hashbrowns. This meal had about 50 grams of carbohydrates 51 g of fat one third of that was saturated fat and 34 g of protein. So that’s about 40 40 20 breakdown of carb fat protein. The second group were given a meal of oatmeal ,milk,, peanut butter, English muffin, raisins and orange juice this meal was 58% carbohydrate 27% fat and 15% protein. Both meals contain the same amount of calories a little over 900. The second lower fat meal was described as being rich in fruit and fibre.
Blood samples were collected at 0,1,2 and three hours. So the-high carbohydrate meal increased glucose significantly at hours one, two and three. The high fruit high fibre meal did not in fact is lowered glucose. Insulin increased in similar manner from both meals as did triglycerides though that was higher in the high-fat high carb meal. Free fatty acids fell after both meals significantly in both but the drop was more significant in the-high fibre meal group.
Measurements of reactive oxygen species were higher at almost all times in the high-fat high carb meal group. NfkB TNF alpha and C-reactive protein were also higher in the high-fat high carb meal.
TLR4 and TLR2 along with SOCS-3 were all significantly increased two and three hours after the high-fat meal. SOCS-3 is seen to be an indicator of an increase in inflammatory cytokines.
Plasma endotoxin and lipopolysaccharide binding protein were both increased by the high-fat high carb meal whereas the high fruit high fibre meal did not induce any significant changes in either.
Other than to say that the effect of a high-fat meal increases endotoxin and all the markers associated with it I’m not sure how much we can take from this. The meals were very complex in terms of the breakdown of elements, macronutrients, types of fat, and fibres. The inclusion of orange juice and minerals may be a more significant factor than fibre which seems to be what the authors are gesturing towards. Orange Juice will significantly ameliorate most of the factors measured in this study, probably due to the effect of the flavonoids hesperedin and naringenin (41) .